Project Summary The general drug discovery process can be described as a large funnel where thousands of compounds are screened for their efficacy and toxicological profile in the pursuit of developing a therapeutic with specific clinical properties. This funnel consists of a battery of biochemical and cellular assays for efficacy and toxicity of compounds. One of the most important screening components of this funnel is the use of inexpensive and high-throughput in vitro cell culture models to evaluate likelihood of efficacy or toxicity in vivo. Accurate in vivo correlation is crucial for in vitro models since failure to eliminate unsuccessful candidate compounds in early screens results in failure in more expensive animal/human trials. Until recently, the majority of in vitro drug screening was conducted on monolayer cell cultures?2D layers of cells are exposed to a compound of interest and evaluated through imaging, flow cytometry, or spectrophotometric assays. While this approach served as the basis for drug discovery for decades, there are notable shortcomings in the ability of the results of these simple and inexpensive assays to translate in vivo. The complex three-dimensional environment in vivo causes differences in response compared to monolayer cell cultures since monolayer models do not possess the diverse array of cells, chemistries, and biochemical environments present in tissues. Because of the well-known limitations with traditional monolayer cell models, pharmaceutical companies have begun to rapidly adopt (23.6% CAGR) 3D in vitro 3D culture models since they have been shown to mimic the environment of in vivo tissues more accurately. However, paradoxically, the current techniques that companies are using to characterize these 3D in vitro models do not fully utilize the 3D data that makes these models an improvement over traditional 2D in vitro models. Even the most comprehensive of high content confocal imaging is limited to visualizing only the surface layers of these models. The purpose of this project is to develop three 3D in vitro cell culture assays that can be offered as a service to pharmaceutical companies for the drug discovery process that characterize the entirety of these models. These assays leverage the patented Visikol HISTO-M? tissue clearing technique which has been shown to improve 3D cell culture characterization by allowing for a 3-fold increase in the amount of cells that can be detected. These assays allow pharmaceutical companies to ask and answer complex mechanistic questions about the efficacy of their compounds which will accelerate the drug discovery process and get therapeutics to patients more quickly.